Pharmaceutical Research

, Volume 34, Issue 2, pp 462–478 | Cite as

Effects of Excipient Interactions on the State of the Freeze-Concentrate and Protein Stability

  • Sampreeti Jena
  • Jacqueline Horn
  • Raj Suryanarayanan
  • Wolfgang Friess
  • Alptekin AksanEmail author
Research Paper



The physical state of excipients in freeze-dried formulations directly affects the stability of the active pharmaceutical ingredient (API). Crystallization of trehalose and mannitol in frozen solutions has been shown to be a function of composition. However, to date a detailed study of the effect of concentrations of the API and other excipients on the crystallinity of mannitol and trehalose in frozen solutions has not been reported.


The crystallinity of mannitol and trehalose in frozen solutions was characterized by Differential Scanning Calorimetry, X-ray diffractometry, and FTIR spectroscopy. The secondary structure of BSA was probed by FTIR, and Circular Dichroism spectroscopy in frozen and thawed solutions, respectively.


Trehalose crystallization was accompanied by unfolding of BSA. BSA delayed and reduced the extent of mannitol and trehalose crystallization. Similar effects were observed upon adding D2O (≥5% w/w) and low concentrations of polysorbate 20 (≤0.2% w/w) with retention of BSA in its native conformation. At high BSA to trehalose mass ratio, the protein could stabilize itself in the frozen state, but unfolded upon thawing.


The API and other excipients, in a concentration-dependent manner, influenced the physical state of the freeze concentrate as well as the stability of the API.


crystallization freeze-drying glass transition mannitol trehalose 



Active pharmaceutical ingredient


Area under curve


Bovine serum albumin


Circular dichroism


Differential scanning calorimetry


Freeze-concentrated liquid


Fourier transform infrared spectroscopy


High performance size exclusion chromatography




Light obscuration


Powder x-ray diffractometry



This research was funded by an NSF grant (CBET-1335936) to A.A. Parts of this work were carried out in the Characterization Facility, University of Minnesota, which receives partial support from NSF through the MRSEC program.

Supplementary material

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Figure S1 (DOCX 144 kb)
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Figure S5 (DOCX 176 kb)
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Table S1 (DOCX 19 kb)


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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Biostabilization Laboratory, Department of Mechanical EngineeringUniversity of MinnesotaMinneapolisUSA
  2. 2.Department of Pharmacy, Pharmaceutical Technology and BiopharmaceuticsLudwig-Maximilians-Universität MünchenMunichGermany
  3. 3.Department of PharmaceuticsUniversity of MinnesotaMinneapolisUSA
  4. 4.The BioTechnology InstituteUniversity of MinnesotaMinneapolisUSA

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